Project Summary Acetaldehyde is both an exogenous carcinogen and endogenous metabolite. While acetaldehyde formed after consumption of alcoholic beverages is well-known as a hepatotoxin and contributor to the developmental abnormalities associated with fetal alcohol syndrome, endogenous production of acetaldehyde was only recently linked to the genetic disease, Fanconi anemia. Acetaldehyde forms DNA adducts, including interstrand DNA crosslinks, repaired by the Fanconi anemia/BRCA pathway. Alcohol metabolism also induces mitochondrial autophagy, or mitophagy, as a cytoprotective mechanism. An entirely new function for Fanconi anemia/BRCA proteins in mitophagy was recently described, highlighting a potential role for the FA/BRCA in responding to acetaldehyde-induced cellular injury. Both Fanconi anemia and fetal alcohol syndrome are characterized by congenital anomalies, and Fanconi anemia and alcohol predispose to an overlapping collection of epithelial neoplasms. Cellular sensitivity to acetaldehyde is poorly understood, and is likely affected by endogenous production of acetaldehyde, metabolism, generation of covalent DNA and protein adducts, and repair pathways. Nave and primed embryonic stem cells represent pre- and post-implantation pluripotent populations in the developing embryo. We demonstrate that nave embryonic stem cells are strikingly tolerant of 1 mM acetaldehyde, while primed embryonic stem cells are exquisitely sensitive (in fact, our collaborator, Dr. Carol Ware, uses acetaldehyde routinely to select against any primed cells that may arise during culture of nave embryonic stem cells). In preliminary data, we show that both types of embryonic stem cell generate acetaldehyde and exhibit basal levels of acetaldehyde adducts with DNA to different degrees. Surprisingly, resistance of nave embryonic stem cells to acetaldehyde is not diminished by inhibition of acetaldehyde-metabolizing aldehyde dehydrogenases. In this proposal, we will investigate the mechanisms of differential acetaldehyde resistance in embryonic stem cells. Specifically, in Aim 1 we will utilize sensitive DNA adductomic profiling to examine the role of the Fanconi anemia/BRCA DNA repair pathway or mitochondrial DNA repair in acetaldehyde resistance. In Aim 2, we will investigate the regulation of the mitophagic response to acetaldehyde by the FA/BRCA pathway. Acetaldehyde inhibits sirtuin 3, the mitochondrial deacetylase, most likely by depleting its NAD+ substrate, leading to a hyperacetylated mitochondrial proteome. Acetylation inhibits mitophagy. We will determine whether the FA/BRCA pathway reduces mitochondrial protein acetylation, thus enabling quality control via mitophagy. The proposed research will generate important new information for understanding cellular responses to acetaldehyde, both in the context of alcohol consumption and genetic syndromes of acetaldehyde hypersensitivity.